Renal cancer accounts for 3% of adult malignancies in the US, and is the 6th leading cause of cancer mortality. In this application, we propose to examine the molecular basis of the oncogenic process leading to the most common form of kidney cancer, renal clear cell carcinoma (RCC). There is a well established link between RCC oncogenesis and the loss of function of the von Hippel-Lindau tumor suppressor protein (pVHL). It is known that this oncogenic effect can be mediated through disruption of HIFa ubiquitylation. However, in this application, we will expand on our previous work implicating a novel pVHL-dependent signaling pathway in the development of RCC: the hydroxylation of the large subunit of RNA Polymerase II (RNAPII), Rpb1, and its subsequent ubiquitylation by the pVHL tumor suppressor complex. Our working hypothesis is that P1465 hydroxylation and pVHL-dependent ubiquitylation of Rpb1 provide fine tuning of the RNAPII complex's transcriptional activity, which results in patterns of gene expression that facilitate appropriate responses to environmental changes (such as oxidative stress). We propose that loss of that fine-tuning results in dysfunctional activity of RNAPII leading to oncogenesis. The overall goal of this work is to understand the mechanism of pVHL-dependent Rpb1 hydroxylation and ubiquitylation, and to assess the role that disruption of this process plays in oncogenesis. To achieve this, we will 1) identify the lysines within ubiquitin and Rpb1 that participate in pVHL-dependent polyubiquitylation of Rpb1 in a P1465-dependent manner in response to oxidative stress; 2) identify the prolyl hydroxylases involved in hydroxylation of Rpb1; 3) determine the oncogenic properties of Rpb1 mutants that do not undergo pVHL-dependent hydroxylation and ubiquitylation; and 4) assess the status of Rpb1 hydroxylation and ubiquitylation in human RCC tumors as compared to normal kidney tissue. The prognosis for patients with RCC is much better if their cancer is diagnosed in early stages; however, early diagnosis is uncommon in RCC because patients often do not experience symptoms until advanced stages of the disease. Because this research explores a novel biochemical mechanism for disease development, it could improve the prognosis for RCC patients in two ways: 1) by contributing to the development of new drugs to treat RCC, and 2) by identifying new genetic markers that could be used to identify individuals at risk for developing RCC.